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Updated: May 28, 2026

Förster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features
Published on: March 16, 2022
Insights into the molecular determinants of EF-G catalyzed translocation
Leyi Wang1, Roger B Altman, Scott C Blanchard
1Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, New York 10065, USA.
Abstract:
Translocation, the directional movement of transfer RNA (tRNA) and messenger RNA (mRNA) substrates on the ribosome during protein synthesis, is regulated by dynamic processes intrinsic to the translating particle. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, in combination with site-directed mutagenesis of the ribosome and tRNA substrates, we show that peptidyl-tRNA within the aminoacyl site of the bacterial pretranslocation complex can adopt distinct hybrid tRNA configurations resulting from uncoupled motions of the 3'-CCA terminus and the tRNA body. As expected for an on-path translocation intermediate, the hybrid configuration where both the 3'-CCA end and body of peptidyl-tRNA have moved in the direction of translocation exhibits dramatically enhanced puromycin reactivity, an increase in the rate at which EF-G engages the ribosome, and accelerated rates of translocation. These findings provide compelling evidence that the substrate for EF-G catalyzed translocation is an intermediate wherein the bodies of both tRNA substrates adopt hybrid positions within the translating ribosome.
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